The present invention relates to vectors, cells and methods useful for making viral eukaryotic gene transfer vectors.
Viral eukaryotic gene transfer vectors have a variety of utilities from the in vitro and in vivo study of cell biology to clinical therapies that alleviate medical conditions. These viral vectors usually must be propagated in a host cell line in vitro prior to use experimentally, clinically, or otherwise. However, some vector designs are not produced efficiently, or are not produced at all, in the host cell line. In some instances, the virus that forms the basis of the viral vector can be so virulent that it efficiently kills the host cells at low or moderate multiplicities of infection (MOI; a low or moderate MOI is an MOI of about 0.1 to about 5 pfu/cell and about 3 to about 20 pfu/cell, respectively) and the viral vector is not effectively replicated in vitro.
In other instances, one or more passenger genes carried by these vectors can cause them to be substantially more cytostatic, cytotoxic, or apoptotic to eukaryotic host cells. In these cases, it has been shown that the passenger gene carried by the viral vector is primarily responsible for the poor production characteristics of these vectors since similar vectors that lack passenger genes are produced efficiently. Moreover, the transduction of eukaryotic cells with viral eukaryotic gene transfer vectors can seriously impede the host cell""s metabolism independently of the passenger gene. When the deleterious effects of the passenger gene act in concert with the deleterious effects of transduction by the gene transfer vector, the cell can be compromised to the extent that few or no gene transfer vectors are produced. Many of the most useful viral eukaryotic gene transfer vectors currently contemplated in the art fit this description.
One result of the transduction of a viral gene transfer vector comprising a deleterious gene into a host cell can be the apoptosis of the host cell. The term xe2x80x9capoptosisxe2x80x9d is well understood in the biological arts and is characterized by a number of phenomena, including xe2x80x9ccytoplasmic boiling,xe2x80x9d severe chromatin condensation and chromosomal fragmentation. It is known that many eukaryotic viruses carry anti-apoptotic genes that facilitate survival of the host cell until viral replication has proceeded to an extent sufficient to ensure the propagation of the virus (e.g., the 19K E1B product of adenoviruses). Nevertheless, viral eukaryotic gene transfer vectors, in particular adenoviral vectors, comprising certain passenger genes lack the capacity to prevent rapid and severe apoptotic responses such that no yield or poor yields of the desired vector are obtained by passaging on typical host cells.
Therefore, there exists a need for a better method of producing cytostatic, cytotoxic, or apoptotic vectors in eukaryotic host cells, particularly those comprising passenger genes that strongly induce these effects. The present invention provides vectors and cells for the production of viral eukaryotic gene transfer vectors that comprise a passenger gene that diminishes the yield of the viral vector because the passenger gene product is cytotoxic or cytostatic, or induces the apoptosis of a host cell for the viral vector. The present invention also provides methods of producing such vectors. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention and examples provided herein.
The present invention provides a method of producing in vitro a viral eukaryotic gene transfer vector, particularly an adenoviral vector, that is deleterious itself, or comprises a deleterious gene, e.g., a cytostatic, cytotoxic, or apoptotic gene. The method comprises inhibiting the deleterious effects of the viral eukaryotic gene transfer vector or the deleterious gene of which it is comprised on eukaryotic, e.g., mammalian, host cells by expressing in the host cells a blocking gene that blocks the deleterious effects of the viral eukaryotic gene transfer vector or the deleterious gene of which it is comprised. The blocking gene can be part of the host cell (e.g., integrated into the host cell genome or present on a plasmid or vector) or the viral eukaryotic gene transfer vector. Accordingly, the present invention also provides novel cells and viral eukaryotic gene transfer vectors. The invention may best be understood in the following detailed description of the preferred embodiments.